The rat pheochromocytoma cell line, PC12, is the premier cell culture model for the differentiating actions neurotrophin, Nerve Growth Factor (NGF). We have proposed a unifying mechanism to explain NGF actions on PC12 cells. Morphological differentiation by NGF is mediated by a proto- oncogene signaling pathway through the sequential activation of Trk, Src, Ras and Raf proteins. Branchpoints off this linear pathway mediate phosphorylation and genetic regulation leading to other neuronal properties, such as neurotransmitter synthesis and the acquisition of electrical excitability. In the next grant period we will test specific predictions of this model and identify critical biochemical intermediates in the NGF pathway to neuronal differentiation. Our specific aims are to: l) test the prediction that Src, Raf and a novel protooncoprotein Shc, mediate NGF-induced differentiation, 2) test the hypothesis that Ras- and Raf-dependent protein kinases are intermediaries in the pathway to stimulation of neurotransmitter synthesis, and 3) examine the mechanism by which Ras and Raf mediate NGF-induction of two neural specific genes, VGF and GAP-43, which are regulated at the transcriptional and post- transcriptional levels, respectively. All of the specific aims rely on the generation of transient and stable PC12 sublines expressing activated or dominant interfering forms of Src, Ras, Raf,, Shc, and MAP kinase. In the vectors used to generate the new PC12 lines, the oncogene or kinase cDNAs will be placed under control of constitutive and/or inducible promoters. In specific aim l, the requirements for and ordering of oncogene activities will be determined by assaying neuronal differentiation in cells expressing combinations of activated and dominant interfering forms of the oncogenes. In specific aim 2, phosphorylation of tyrosine hydroxylase (TH) will be used to assay for kinases activated in the NGF signaling pathway. In PC12 sublines expressing activated or dominant interfering forms of Ras, Raf, or MAP kinase, Th phosphorylation will be analyzed by phosphopeptide mapping. FPLC technology will be used to purify a novel Raf-dependent kinase activity which mediates long term phosphorylation of TH on serine 31. In specific aim 3, the PC12 sublines expressing activated or dominant interfering forms of Ras and Raf, will be host cells for transfections by plasmids containing regulatory fragments of the VGF and GAP-43 genes. Mutational analysis will be used to determine those gene sequences which confer regulation. The proposed studies will have direct relevance to the entire family of neurotrophins, which regulate neuronal survival and differentiation in vivo. These approaches are expected to yield insights into the possible causes of the loss of critical neuronal properties, as well as rational approaches to treatment in Parkinson's, Alzheimer's, familial dysautonomic, and other related central and peripheral neuron degenerating diseases.